Styrene is a useful and versatile monomer for the production of numerous polymers and co-polymers, which accounts for 60% of its total global use1. Styrene is most commonly yielded by the chemocatalytic dehydrogenation of petroleum-derived ethylbenzene (U.S. Pat. No. 4,255,599), a process requiring over 3 metric tons of steam per metric ton of styrene produced. This exorbitant requirement renders styrene production as the most energy-intensive among commodity chemical production routes, consuming nearly 200 trillion BTU of steam for its domestic annual production alone2. In 2006, over 6 million metric tons of styrene were produced by U.S. manufacturers alone, representing a market that is currently valued at nearly $28 billion and projected to grow by 4.3% per year through at least 20101. A more sustainable and inexpensive approach would involve the engineering of microorganisms that possess the unique ability to synthesize styrene at high levels directly from renewable resources. Presently, however, an inexpensive and sustainable source of styrene has not yet been developed.
A variety of additional novel synthetic routes have recently been engineered in microorganisms for the production, from substrates such as glucose, of a number of other useful monoaromatic compounds with structural similarity to styrene. For example, a biosynthetic pathway for the production of p-hydroxystyrene (pHS; a monomer used in polymer synthesis) from renewable sugars has been reported using E. coli4 or P. putida5 as the engineered host platform. Meanwhile, both phenol (a precursor and monomer for phenolic resins)6 and p-hydroxybenzoate (a precursor to parabens, which are used as preservatives)7 have also been synthesized as individual products from glucose by engineered strains of P. putida. These studies further illustrate how, through metabolic engineering strategies, microbial biocatalysts can be developed for the sustainable biosynthesis of a variety of important commodity chemicals of monoaromatic nature from renewable resources. Each of the above non-natural metabolites were derived using L-tyrosine (or its immediate precursor, 4-hydroxyphenylpyruvate) as a precursor (thereby making them all phenolics). There are, however, no previously reported studies on the production of styrene from renewable resources (for example, carbohydrates such as glucose) by either naturally-occurring or recombinant microorganisms.
L-Phenylalanine is a naturally-occurring, proteinogenic amino acid that is ubiquitous among most all living organisms. Although its natural biosynthesis is often tightly regulated, its overproduction on fermentable sugars has been engineered in several microorganisms, and most notably in Escherichia coli (U.S. Pat. No. 4,681,852) and Corynebacterium glutamicum (U.S. Pat. No. 3,660,235).
Phenylalanine ammonia lyase (PAL) activity has been reported in a number of marine bacteria, including Anabaena variabilis, Nostoc punctiforme, and Streptomyces maritimus, and the genes have been identified8-10. In addition, the yeast Rhodotoruloides glutinis has been well-studied with regards to its phenylalanine ammonia lyase (PAL) activity, however, the identified and characterized gene product is less specific in that it also functions as a tyrosine ammonia lyase (TAL)4, 11. It is further known that the yeast Saccharomyces cerevisiae is capable of synthesizing styrene when supplied with exogenous trans-cinnamic acid (Calif.)12. That is to say, the yeast Saccharomyces cerevisiae is known to naturally display trans-cinnamic acid decarboxylase (CADC) activity. It has been further demonstrated that this native enzymatic ability has an essential dependence on the combined expression of the enzymes encoded by the genes PAD1 and FDC113.
In light of the foregoing, it would be an advancement in the current state of the art to provide a method by which styrene could be produced from inexpensive and sustainable resources such as carbohydrates or sugars. It would be particularly advantageous if the method produced a high level of styrene at high substrate yields and with a limited diversity and quantity of by-products. The development of such a method will require the ability to manipulate and assemble the appropriate genetic machinery responsible for the conversion of carbohydrates such as glucose to CA, and CA to styrene. It would be exceptionally advantageous if these conversions could all be achieved within a single host cell.
The above mentioned biological and chemical systems provide both examples of a number of potentially useful genetic elements, as well as a number of pathways that may be useful in the biological production of styrene, however the efficient biological production of styrene has not been achieved. Therefore, the problem to be overcome is to design and develop a method for the efficient production of styrene by a biological source using inexpensive substrates as the carbon source. The applicants have solved the stated problem by engineering a microbial host to produce styrene by expression of foreign genes which encode phenylalanine ammonia lyase (PAL) and trans-cinnamic acid decarboxylase (CADC).
Furthermore, (S)-styrene oxide may be produced by the enzymatic oxidation of styrene by the additional co-expression of a gene encoding a polypeptide with styrene monooxygenase (SMO) activity. Epoxides are desirable compounds due to their versatile nature as chemical building blocks. More specifically, (S)-styrene oxide is a functional building block that is used as a precursor to a variety of pharmaceutical compounds including levamisole and some analgesics14. Enzymatic reactions, as opposed to chemical processes, have the unique ability to yield enantiomerically pure products. The styrene oxygenase activity of several Pseudomonas sp. has been identified as the two-component styrene monooxygenase encoded by styAB14. Activity has also been reported for the two-component flavoprotein monooxygenase encoded by styA2B present in Rhodococcus opacus 1CP15.